The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5 extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans Ϸ25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

Proteins anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) moiety are found in all eukaryotes. After NH2-terminal peptide cleavage of the nascent protein by the signal peptidase, a second COOH-terminal signal peptide is cleaved with the concomitant addition of the GPI unit. The proposed mechanism of the GPI transfer is a transamidation reaction that involves the formation of an activated carbonyl intermediate (enzyme-substrate complex) with the ethanolamine moiety of the preassembled GPI unit serving as a nucleophile. Other nucleophilic acceptors like hydrazine (HDZ) and hydroxylamine have been shown to be possible alternate substrates for GPI. Since GPI has yet to be purified, the use of readily available nucleophilic substitutes such as HDZ and hydroxylamine is a viable alternative to study COOH-terminal processing by the putative transamidase. As a first step in developing a soluble system to study this process, we have examined the amino acid requirements at the COOH terminus for the transamidation reaction using HDZ as the nucleophilic acceptor instead of GPI. The hydrazideforming reaction shows identical amino acid requirement profiles to that of GPI anchor addition. Additionally, we have studied other parameters relating to the kinetics of the transamidation reaction in the context of rough microsomal membranes. The findings with HDZ provide further evidence for the transamidase nature of the enzyme and also provide a starting point for development of a soluble assay.Glycosylphosphatidylinositol (GPI) anchoring is a mechanism used by all eukaryotic cells to tether proteins to the outer cell membrane (1-7). A nascent protein (preproprotein) that is destined to be GPI anchored contains an NH2-terminal signal sequence and a COOH-terminal signal sequence, both of which are cleaved sequentially first by the NH2-terminal signal peptidase and then by a putative transamidase to yield the mature GPI-linked protein. Cleavage at the COOH-terminal signal proceeds in a concerted manner in which the GPI moiety is incorporated at the newly exposed COOH terminus of the protein.The amino acid residue that condenses with the GPI moiety is designated the X residue (8,9). Analogous to the -1, -3 rule that has been described for NH2-terminal signal peptidase activity, an wc,w+2 rule for COOH-terminal processing has been described (8). (8) (Fig. 1A).In previous work with an in vitro translation system employing rough microsomal membranes (RM) (8), we demonstrated that the proform of an engineered version of human placental alkaline phosphatase (PLAP), miniPLAP, is processed to the mature COOH-terminal cleaved form. Processing of nascent preprominiPLAP (28 kDa) gives rise to prominiPLAP (27 kDa), in which the NH2-terminal signal sequence is removed. This proform is subsequently converted to mature miniPLAP (24.7 kDa), in which a 29-residue COOH-terminal signal sequence is cleaved and replaced by GPI. In addition to the formation of this anchor-containing product (Fig. 1B), cleavage of prominiPLAP...

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitationcontraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b)they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode at 70 ms (slow gating); a small number of openings had short first latencies, <12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from "fin = 110 ms (reference) to T,,~ = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six-state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltagedependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating modes of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels.